The Notch receptor family is a class of evolutionarily conserved transmembrane receptors that transmit signals affecting development in organisms as diverse as sea urchins and humans. Both Notch receptors and its family of ligands Delta and Serrate (known as Jagged in mammals) are transmembrane proteins with large extracellular domains that contain epidermal growth factor (EGF)-like repeats. The number of Notch paralogues differs between species. For example, there are four Notch receptors in mammals (Notch1-Notch4), two in Caenorhabditis elegans (LIN-12 and GLP-1) and one in Drosophila melanogaster (Notch). Notch receptors are proteolytically processed during transport to the cell surface by a furin-like protease at a site S1 external to the transmembrane domain, producing an extracellular Notch (ECN) subunit and a Notch transmembrane subunit (NTM). These two subunits remain non-covalently associated and constitute the mature heterodimeric cell-surface receptor. Notch1 ECN subunits contains 36 N-terminal EGF-like repeats followed by three tandemly repeated Lin 12/Notch Repeat (LNR) modules that precede the S1 site. Each LNR module contains three disulfide bonds and a group of conserved acidic and polar residues predicted to coordinate a calcium ion. Within the EGF repeat region lie binding sites for the activating ligands. The LNR modules, which comprise a unique domain of Notch receptors, participate in maintaining Notch in a resting conformation before ligand-induced activation. The Notch1 NTM comprises an extracellular region (which harbors the S2 cleavage site), a transmembrane segment (which harbors the S3 cleavage site), and a large intracellular part that includes a RAM domain, ankyrin repeats, a transactivation domain and a carboxy-terminal PEST sequence. Stable association of the ECN and NTM subunits is dependent on a heterodimerization domain (HD) comprising the carboxy-terminal end of the ECN (termed HD-C) and the extracellular amino-terminal end of NTM (termed HD-N). Binding of a Notch ligand to the ECN subunit initiates two successive proteolytic cleavages that occur through regulated intramembrane proteolysis. The first cleavage by a metalloprotease at site S2 renders the Notch transmembrane subunit susceptible to the second cleavage at site S3 close to the inner leaflet of the plasma membrane. Site S3 cleavage, which is catalyzed by a multiprotein complex containing presenilin and nicastrin, liberates the intracellular portion of the Notch transmembrane subunit, allowing it to translocate to the nucleus and activate transcription of target genes.
Five Notch ligands of the Jagged and Delta-like classes have been identified in humans (Jagged1 (also termed Serrate 1), Jagged2 (also termed Serrate2), Delta-like1 (also termed DLL1), Delta-like3 (also termed DLL3), and Delta-like4 (also termed DLL4)). Each of the ligands is a single-pass transmembrane protein with a conserved N-terminal Delta, Serrate, LAG-2 (DSL) motif essential for binding Notch. A series of EGF-like modules C-terminal to the DSL motif precede the membrane-spanning segment. Unlike the Notch receptors, the ligands have short cytoplasmic tails of 70-215 amino acids at the C-terminus. In addition, other types of ligands have been reported (e.g., DNER, NB3, and F3/Contactin).
The Notch pathway functions during diverse developmental and physiological processes including those affecting neurogenesis in flies and vertebrates. In general, Notch signaling is involved in lateral inhibition, lineage decisions, and the establishment of boundaries between groups of cells (see, e.g., Bray, Molecular Cell Biology 7:678-679, 2006). A variety of human diseases, including cancers and neurodegenerative disorders have been shown to result from mutations in genes encoding Notch receptors or their ligands (see, e.g., Nam et al., Curr. Opin. Chem. Biol. 6:501-509, 2002). The connection between unrestrained Notch signaling and malignancy was first recognized when a recurrent t(7;9)(q34;q34.3) chromosomal translocation which creates a truncated, constitutively active variant of human Notch1 was identified in a subset of human acute lymphoblastic leukemias (T-ALL). In mouse models, Notch1 signaling has been shown to be essential for T cell development and that Notch1-mediated signals promote T cell development at the expense of B cell development. Also, in mouse models, excess Notch signaling during development leads to T cell neoplasia.
Moreover, Notch receptors are expressed in a wide-range of human cancers and tumor-derived cell lines and promote neural fates in human embryonic stem cells. For instance, Notch is highly expressed in neoplastic lesions in the human cervix and in human renal cell carcinoma cells. Given the involvement of Notch signaling in a wide variety of human disease it is clear that there continues to be a need for agents that regulate Notch signaling that have clinical attributes that are optimal for development as therapeutic agents. The invention described herein meets this need and provides other benefits.
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